Method for regulating the expression of a gene in a baculovirus using a retinoic acid receptor binding site and vector therefor

ABSTRACT

A recombinant vector useful for regulating the expression of a gene  contred by a baculovirus polyhedrin promoter or P10 promoter, via a sequence forming an RAR-type hormone receptor binding site.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to new expression vectors obtained frombaculoviruses.

DESCRIPTION OF THE BACKGROUND

The baculoviruses, represented by the Autographa californica nuclearpolyhedrosis virus (AcMNPV), possess several promoters which are activein different phases of the viral replication cycle. Some of thesepromoters are used in genetic engineering to control the expression ofheterologous genes inserted into the baculovirus genome. Among the onesmost commonly employed, two strong very late promoters may be mentioned:the polyhedrin (polh) promoter and the P10 polypeptide promoter, whichare active only at the end of the infectious cycle, after replication ofthe viral genome, and which enable the genes placed under their controlto be expressed at a high level.

The polh and P10 promoters are described in detail in the followingpublications: POSSEE and HOWARD Nucleic Acid Research, vol. 15, pp.10233-10248 (1987)! for the polyhedrin promoter, and QIN et al. J. Gen.Virol. vol. 70, pp. 1273-1279, (1989)! for the P10 promoter. Thesequence localized between positions (-71 and +1) defined relative tothe A(+1) of the polyhedrin ATG is generally defined as the "polyhedrinpromoter", and the sequence localized between positions (-70 and +1)defined relative to the A(+1) of the p10 polypeptide ATG is generallydefined as the "p10 promoter".

The obtaining from a baculovirus, of a vector expressing a foreign geneunder transcriptional control of a promoter of the baculovirus, isgenerally performed by methods known per se, through construction of atransfer vector containing the the promoter, then recombination with theDNA of the wild-type virus, and lastly selection of the recombinants.

SUMMARY OF THE INVENTION

During the replication cycle of the baculovirus, the early genes areexpressed first: the transcription of these genes involves the hostcell's RNA polymerase II.

Subsequently, the late and very late genes such as polh and P10 aretranscribed by a particular RNA polymerase, of at least partially viralorigin, which is insensitive to α-amanitin.

An A/GTAAG motif common to all the late genes constitutes thetranscription startsite. This motif is essential for the recognition ofthese promoters by the RNA polymerase, and hence for their activity. Themechanisms which govern the transcriptional activation are not yet,however, precisely known.

Previous work of the inventors' team has shown that it is especiallyadvantageous, in order to increase the level of expression of aheterologous gene placed under the control of one of the two strong verylate promoters polh or P10, to construct a baculovirus in which only oneof these two strong late promoters is inactivated, and to cause the saidgene to be expressed under the control of the remaining promoter.

This principle has been used as a basis for the construction of severalmodified baculoviruses European Application No. 91 913 605.1 in thenames of the INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE (I.N.R.A.)and of the CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.);CHAABIHI et al., J. Virol., 67, 2664-2671 (1993)!, in which either theP10 promoter (baculovirus designated AcSLP33, for example) or thepolyhedrin promoter (baculovirus designated AcSLP10, for example) isinactivated by deletion.

In the context of continuing their work on the regulation of expressionin baculoviruses, it occurred to the inventors to study the influence ofthe coding sequence of the P10 gene on the expression of the latter. Forthis purpose, they made, from the wild-type baculovirus AcMNPV,constructs comprising the CAT reporter gene inserted at one of thepositions +16, +150 or +230 of the sequence coding for 10 (thesepositions are defined relative to the A (+1) of the ATG initiation codonof P10), and studied the simultaneous expression of the CAT reportergene and of the polyhedrin gene in the recombinant viruses obtained(designated AcCAT+16, AcCAT+150 and AcCAT+230, respectively), relativeto the wild-type baculovirus AcMNPV and to the modified baculovirusAcSLP33 (in which the P10 promoter is inactivated).

The recombinant viruses AcCAT+16, AcCAT+150 and AcCAT+230 are showndiagrammatically in FIG. 1.

This allowed the inventors to notice that when the reporter gene isinserted at +16 (this insertion is accompanied by the deletion of aportion of the sequence coding for P10), the expression of the reportergene is weak, while the expression of the polyhedrin gene increases verysignificantly although the P10 promoter is intact, until reach a levelcomparable to the one observed in a virus in which the P10 promoter isinactivated, such as the virus AcSLP33.

In contrast, when the reporter gene is inserted further downstream (+150or +230), the expression of the said gene increases, and that ofpolyhedrin decreases to return to a baseline level comparable to thatobserved in the wild-type baculovirus AcMNPV.

Moreover, during other investigations under taken independently, theinventors sought to express the α and γ isoforms of the human retinoicacid receptor (hRAR) in insect cells, and constructed for this purpose,from wild-type AcMNPV baculoviruses, recombinant baculoviruses whichexpress the said α or γ iso-forms, respectively, under the control ofthe polyhedrin promoter. Now, during these investigations, they observedan unexpected overexpression of the P10 gene in these baculoviruses.

This activation is revealed by the mRNA level which is twice as high asthe control (consisting of the wild-type baculovirus AcMNPV) in the caseof the α receptor, and 5 times as high in the case of the γ receptor:the activation is hence taking place at the transcriptional level.

Moreover, this activation is observed not only during the transfectionof a cell with a double-recombinant virus, but also during thecotransfection of a cell with two plasmids (one expressing an RAR andthe other carrying an RARE domain localized in the P10 gene); theactivation is hence a trans-activation.

The inventors established a parallel between the results of the twoseries of investigations reported above, and went on to analyse thesequence of the P10 region in order to look for the possible existenceof sequences related to the ones constituting binding sites for nuclearreceptors, and especially for retinoic receptors.

The retinoic acid receptors (RARs) belong to the family of "nuclearreceptors" which also comprises, for example, in vertebrates, thevitamin D3 receptors or the thyroid receptors. Other receptors,designated RXRs and which are, like the RARs, activated by retinoic acidor by other retinoids, have also been described.

Nuclear receptors are known to be capable of activating thetranscription of a target gene. This activation involves, on the onehand the binding of these nuclear receptors to their specific ligand,and on the other hand their binding to DNA, which involves therecognition of short consensus DNA sequences known as "responseelements" generally situated upstream of the target gene. The RARs andRXRs thus recognize consensus sequences which are repeated once orseveral times, in tandem or palindromically, and define portions of DNAknown as RARE or RXRE elements, respectively GORDMAN et al. Mol. Cell.Biol. 2, 1044-1051 (1982)!. Various consensus sequences have beenidentified. For example, the response elements for the RARβ receptor,which are described in Application PCT W091/07488, comprise tandemrepeats of the sequence GTTCAC; Application PCT WO 92/16658 describesRXRE response elements, and especially an RXRE element obtained from thepromoter of the rat CRBPII gene, which comprises tandem repeats of thesequence AGGTCA.

The response elements for the nuclear receptors have a variablespecificity. Some, like the thyroid hormone receptor response element(TRE), respond not only so their own receptor but also to FARs and toRXRs. Others are more specific, like the PARβ receptor response elementswhich respond only weakly to RXRs. Others, like the RXRE element whichis described in Application PCT WO 92/16658, can bind RAR or RXRreceptors, but only the latter produce a response which brings about theactivation of the promoter placed downstream of this RXRE element. Thistrans-activation by RXRs is blocked in the presence of RARs.

In insects, there are also proteins akin to RARs; particular examplesare the hormone receptor for ecdysone (EcR) and for the USP protein ofDrosophila. Application PCT WO 92/14695 describes an RXR type receptor,designated XR2C, obtained from Drosophila.

The proposal has been made to place response elements for RARs or forRXRs upstream of the promoter of a gene, at a distance from the saidpromoter of between 30 bp and 10,000 bp, so as to obtain the activationof the said gene via RAR or RXR receptors expressed in the same cell.For example, Application P4T WO 91/07488 proposes the use of RARPreceptor response elements for activating the transcription of genes inmammalian cells. Application PCT WO 92/16658 proposes the use of theRXRE response elements which it describes for studying thetrans-activation of genes by RXR receptors, and the blocking of thistransactivation in the presence of XRA receptors in mammalian, bird orinsect cells. Application PCT WO 92/14695 mentions several responseelements which are considered to be usable with the XR2C receptor; infact, it describes the use of this receptor only with a TRE responseelement placed upstream of the ADH promoter of Drosophiia.

Analysis of the sequence of the P10 region, performed by the inventors,revealed the existence in the P10 gene of an intragenic sequenceGTTGACAGTGTTCA (SEQ ID NO:2), similar to a consensus sequence recognizedby RARs and hence constituting a putative RARE element. This sequence islocalized, in wild-type AcMNPV, at position +61 relative to the A(+1) ofthe ATG initiation codon of P10.

Further experiments performed by the inventors have enabled it to beshown that activation of the P10 gene, or of a reporter gene placedunder the transcriptional control of the P10 promoter, in the presenceof the product of an RAR gene placed under the control of the polyhedrinpromoter, takes place on condition that the RARE type domain localizedin the P10 gene is present. In addition, this activation also takesplace when the RARE type domain is removed from its initial position andput back in another position in proximity to the P10 promoter,downstream or upstream of this promoter. Lastly, if the RARE type domainis placed in the same manner in proximity to another promoter, theactivation of this other promoter is observed. In all cases, this RAREelement is active irrespective of whether it i placed upstream ordownstream of the promoter, and regardless of its orientation withrespect to the latter.

Analysis of the sequence of the P10 gene in the baculovirusesChoristoneura fumiferana NPV and Bombyx mori NPV reveals theconservation of the RARE motif. Hence these baculoviruses can, on thesame basis as AcMNPV, be used for carrying out the present invention.

The finding by the inventors of the RARE-like intragenic sequence and ofits effective role in the regulation of the expression of the P10 geneallow to provide new means of regulating the expression of heterologousgenes coding for proteins of interest under the control of baculoviruspromoters.

The present invention relates to these means of regulation, whichinclude several variants.

According to a first variant of the invention, the activation of abaculovirus promoter in a host cell, especially of a strong latepromoter such as the P10 or polh promoter, is obtained in the presenceof the product of an RAR gene in the same host cell, and of an RARE typesequence situated in cis and in proximity to the said baculoviruspromoter.

For the purposes of the present invention an RARE sequence is consideredto be "in proximity to a promoter" if it lacuna! situated downstream orupstream of the said promoter at a distance of between 10 and 10,000 bp,and preferably less than 1000 bp, from the transcription startsite ofthe said promoter.

The subject of the present invention is a method for regulating theexpression of a gene placed under the transcriptional control of abaculovirus promoter in an expression vector comprising an RARE typesequence situated in proximity to the said baculovirus promoter, whichmethod is characterized in that the expression of the said gene iseffected in the presence of the translation product of a gene coding foran RAR type hormone receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: illustration of transfer vectors.

FIG. 2: a map of the p10 region.

FIG. 3: illustration of the plasmid inserts pMH16, pMH150 and pMH230.

FIG. 4: (A) and (B), illustration of recombinant viruses.

DETAILED DESCRIPTION OF THE INVENTION

The baculovirus promoter can be, for example, the P10 promoter, the polhpromoter, one of the promoters of the IE1 and IEN genes or a syntheticcromoter. Advantageously, it is a strong late promoter such as the P10promoter or the polh promoter.

Preferably, the PAR type hormone receptor is an RARα receptor, an RARβreceptor or an RARγ receptor, and the RARE DNA sequence is a sequence(identified in the attached sequence listing under the number SEQ IDNO: 1) corresponding to the general formula GTTGANNNNGTTCA(SEQ ID NO:3)where N represents A or C or G or T, and especially the sequenceGTTGACAGTGTTCA identified in the attached sequence listing under thenumber SEQ ID NO: 2.

According to a preferred embodiment of a method according to theinvention, the expression of the gene placed under the transcriptionalcontrol of the said baculovirus promoter is effected in the presence, inthe same cell, of a gene expressing an RAR type hormone receptor.

The baculovirus promoter and the gene expressing an PAR type hormonereceptor can be carried by the same DNA molecule, or alternatively bytwo different DNA molecules.

The gene expressing an PAR type hormone receptor may be placed under thecontrol of any promoter, provided the said promoter is expressed in thehost cell. Advantageously, it is placed under the control of a secondbaculovirus promoter; this second promoter and the one under the controlof which the gene of interest is expressed may be identical ordifferent.

To carry out a method according to the invention, it is possible, forexample, to use expression vectors, known per se, which do not carry agene expressing an RAR type hormone receptor, and which comprise atleast the portion of the P10 gene consisting of the promoter of the saidgene followed by the sequence portion coding for the P10 protein whichcomprises the sequence GTTGACAGTGTTCA(SEQ ID NO:2), the said promoterand said coding sequence being arranged in a manner identical to theirarrangement in the wild-type P10 gene. These vectors can be used in thepresence, in the same host cell, of another DNA molecule expressing anRAR type hormone receptor.

New recombinant vectors, which form part of the subject of the presentinvention, can also be used.

Recombinant vectors according to the invention, which can be used forcarrying out a method in which the baculovirus promoter and the geneexpressing an RAR type hormone receptor are carried by two DNAmolecules, consist of recombinant baculoviruses comprising a DNAsequence constituting an RAR type hormone receptor binding site, placedin proximity to a baculovirus promoter under the control of which it isdesired to express a gene coding for a protein of interest, on conditionthat, if the baculovirus promoter is the P10 promoter and the RAREsequence is the sequence GTTGACAGTGTTCA(SEQ ID NO:2), the said sequenceis situated relative to the said promoter in a different location fromthe one it occupies in the wild-type P10 gene, or is separated from thesaid promoter by a sequence different from the one which separates itfrom the wild-type P10 promoter.

GTTGACAGTCTTCA(SEQ ID NO:2) the said sequence is situated relative tothe said promoter in a different location from the one it occupies inthe wild-type P10 gene, or is separated from the said promoter by asequence different from the one which separates it from the wild-typeP10 promoter.

Other recombinant vectors according to the invention, which can be usedfor carrying out a method in which the baculovirus promoter and the geneexpressing an RAR type hormone receptor are carried by the same DNAmolecule, consist of recombinant baculoviruses comprising an RARE DNAsequence constituting an RAR type hormone receptor binding site, placedin proximity to a first baculovirus promoter under the control of whichit is desired to express a gene coding for a protein of interest, and aDNA sequence coding for an PAR type hormone receptor, placed under thetranscriptional control of a second baculovirus promoter.

The first and the second promoter can, for example, be the P10 promoter,the polh promoter, the promoters of the IE1 and IEN genes or syntheticpromoters. A copy of the first promoter can also be used as secondpromoter.

Advantageously, the first promoter is the P10 promoter and the secondpromoter the polh promoter, or alternatively a second copy of the P10promoter. Advantageously also, the first promoter is the polh promoterand the second promoter the P10 promoter, or alternatively a second copyof the polh promoter.

Preferably, the RAR type hormone receptor is an RARα receptor, an RARβreceptor or an RARγ receptor, and the RARE DNA sequence is a sequence asdefined in the attached sequence listing under the number SEQ ID NO:1 orSEQ ID NO:2.

Advantageously, the RARE DNA sequence is placed downstream of the firstpromoter.

According to a preferred embodiment, these vectors comprise, inaddition, at least one heterologous sequence coding for the protein ofinterest which it is desired to express, placed under thetranscriptional control of the first promoter, or at least one site forinsertion of the said sequence.

Vectors according to the invention are, for example, thedouble-recombinant viruses obtained from the recombinant viruses Ac+150and Ac+230, described above, by insertion of a gene coding for one ofthe retinoic acid receptors RARα or RARγ, under the control of thepolyhedrin promoter.

According to a second variant of the invention, an activation of thepolh promoter similar to that observed in expression vectors in whichthe P10 promoter is inactivated is obtained using expression vectorsbereft of the whole of the sequence coding for the P10 protein.

In accordance with this second variant, the subject of the presentinvention is recombinant vectors which can be used to increase theexpression of a gene under the transcriptional control of thebaculovirus polyhedrin promoter, and which consist of modifiedbaculoviruses comprising a polyhedrin promoter and a P10 promoter whichare intact and functional, and in which the region in the vicinity ofthe P10 is bereft of any sequence constituting an RAR type hormonereceptor binding site.

According to a preferred embodiment of the vectors according to thissecond variant of the invention, they are bereft of the whole of thesequence coding for the P10 protein.

According to another preferred embodiment of the vectors according tothis variant of the invention, they comprise, in addition, at least onesequence coding for a heterologous protein which it is desired toexpress, placed under the transcriptional control of a polyhedrinpromoter, or at least one site for insertion of the said sequence.

Vectors according to this second variant of the invention can, forexample, be obtained from an Ac+16 recombinant virus.

According to another preferred embodiment of the vectors according tothis second variant of the invention, they comprise, in addition, a DNAsequence coding for an RAR type hormone receptor, which sequence isplaced under the transcriptional control of a baculovirus promoter otherthan the P10 promoter.

In effect, the inventors observed that, when a gene (such as, forexample, the CAT reporter gene) is inserted under the transcriptionalcontrol of the P10 promoter, and in the absence of the RARE sequence,not only is an absence of activation noted, but even an inhibition ofits expression during coexpression with RARs.

Vectors of this type may be obtained from Ac+16 by insertion of a genecoding for one of the retinoic acid receptors RARα or RARγ, under thecontrol of the polyhedrin promoter; sic!

Vectors according to the invention can constitute transfer vectors orexpression vectors. They may be obtained from any baculovirus orconstruct (such as a transfer vector) derived from baculoviruses, oncondition that the said baculovirus or said construct comprisessequences constituting the P10 promoter as are defined above.

A better understanding of the present invention will be gained from thefurther description which follows, which relates to examples ofconstruction and of use of expression vectors according to theinvention.

It should, however, be clearly understood that these examples are givenonly by way of illustration of the subject of the invention and in noway constitute a limitation of the latter.

The protocols used in the examples which follow make use of standardtechniques of genetic engineering, such as the ones described bySAMBROOK et al. Molecular cloning: A Laboratory Manual; Second Edition,Cold Spring Harbor Laboratory, 1989!, or by O'REILLY et al. BaculovirusExpression Vectors: A Laboratory Manual; Freeman and Co., New York,(1992)!, for the manipulation of baculovirus DNA. The conditionsdistinctive to each experiment are, if necessary, specified in thecorresponding examples.

EXAMPLE 1 1) Plasmid DNAs and transfer vectors

A series of three transfer vectors, designated pMH16, pMH150 and pMH230,respectively, was constructed in order to introduce the bacterial genecoding for CAT (chloramphenicol acetyltransferase) into the sequencecoding for P10, at positions +16, +150 and +230.

FIG. 2 depicts a map of the P10 region, showing the positions inquestion.

In these three constructions, the ATG codon of P10 has been mutated toAGC, and a PvuII restriction site was created (sequence ATG TCA mutatedto AGC TGA).

pMH16

The plasmid pMH16 is derived from the transfer vector pGm16. The vectorpGm16 contains an insert obtained from the EcoRI-P fragment of thebaculovirus GmMNPV, mutated as indicated above at the translationinitiation site of P10, and in which the sequence included between thebases +16 and +265 of the P10 gene has been deleted and replaced by aBglII linker.

To obtain pMH16, a 1900 bp HindIII-NsiI fragment isolated from theHindIII-Q fragment of AcMNPV, which contain the hr5 sequences and thep26 gene, was introduced between the HindIII and NsiI sites of pGm16.

The 790-bp BglII-BanI fragment obtained from the plasmid pBLCAT2 (LUCKOWand SCHUTZ, Nucleic Acid Res., 15, (13) 5940 (1987) lacuna! andcomprising the sequence coding for CAT was then introduced into theplasmid at the BglII site.

pMH150 and pMH230

The plasmids pMH150 and pMH230 are derived from the same construct,which was made by inserting a 1900-bp NsiI-EcoRI fragment carryingmutations indicated above of the EcoRI-P fragment of AcMNPV between theNsiI and KpnI sites of the HindIII-Q fragment of AcMNPV, previouslycloned to the vector pUC18.

To obtain plasmid pMH15O, the BglII-BanI fragment of pBLCAT2 comprisingthe sequence coding for CAT was introduced at the BglII site situated atposition +150 in the sequence coding for P10. To obtain plasmid pMH230,the said BglII-BanI fragment was introduced at the HindIII site situatedat position +230 in the sequence coding for P10.

FIG. 3 depicts diagrammatically the inserts of plasmids pMH16, pMH150and pMH230.

Plasmid pPH-RARα and pPH-RARγ

A 1518-bp KpnI-StuI fragment, or a 1564-bp NcoI-SmaI fragment,comprising the sequences in their entirety coding for the elements hPARαPETROVITCH et al., Nature, vol. 330, pp. 444-450 (1987)! and hRARγBENBROOK et al., Nature, vol. 333, pp. 669-672 (1988)!, respectively,were introduced downstream of the polyhedrin promoter into the SmaI siteof the plasmid pGmAc34T DAVRINCHE et al. Biochem. Biophys. Res. Com.,vol 195, pp. 469-477!.

Plasmid pRARα (P10) and pRARγ (P10)

In the same manner, the sequences coding for the hRAR elements wereintroduced at the BglII site of plasmid pMH16.

2) Obtaining of recombinant baculoviruses

For each of the transfer vectors described above, 4×10⁶ Sf9 cells arecotransfected by lipofection (DOTAP, BOEHRINGER MANNHEIM) with 10 μg ofvector and 1 μg of viral DNA.

Recombinations at the polyhedrin site are performed by cotransfectingthe plasmid in question with wild-type AcMNPV virus DNA, whilerecombinations at the P10 site are performed by cotransfecting theplasmid in question with the DNA of a modified baculovirus (AcSLP10) inwhich the promoter and the polyhedrin gene have been excised and thesequence coding for polyhedrin put back under the control of the P10promoter.

Two clones of each recombinant were purified independently.

FIG. 4 depicts diagrammatically the various recombinant virusesobtained. The promoters (P10 and polyhedrin) are represented by a solidblack rectangle, the P10 sequence is represented by a completely clearrectangle, the sequence coding for polyhedrin is represented by aclosely dotted rectangle (), the sequences coding for the RARs arerepresented by a more loosely dotted rectangle () and the sequencecoding for CAT is represented by a hatched rectangle.

FIG. 4A depicts, from top to bottom:

the wild-type AcMNPV virus;

a virus bereft of the polyhedrin promoter and gene (AcD3);

a virus obtained after recombination of a plasmid pPH-RAR with thewild-type virus DNA (AcRARP10);

a virus obtained after recombination of a plasmid pPH-RAR with the DNAof virus bereft of the P10 promoter and gene (AcRARΔP10);

a virus obtained after recombination of a plasmid pRAR(P10) with the DNAof virus bereft of the polyhedrin promoter (AcSLP10RAR);

a virus obtained after recombination of a plasmid pRAR(P10) with thewild-type virus DNA (AcPHRAR).

FIG. 4B depicts diagrammatically the recombinants carrying the CAT geneat different positions within the P10 sequence (+16, -150 and +230), andthe RARα or γ gene at the polyhedrin locus:

a virus obtained after recombination of one of the plasmids pMH16,pMH150 or pMH230 with the wild-type virus DNA (AcP10CAT);

a virus obtained after recombination of a plasmid pPH-RAR with the virusAcP10CAT DNA (AcRARCAT); sic!

EXAMPLE 2 EXPRESSION OF P10 IN THE RAR RECOMBINANTS

The cells (Spodoptera frugiperda Sf9) are maintained at 28° C. in TC100medium (GIBCO/BRL) supplemented with 5% of heat-inactivated foetal calfserum.

The cells are infected with a viral suspension (wild-type AcMNPV or testrecombinant) with a multiplicity of infection of 10 PFU (plage sic!forming units) per cell. After one hour of adsorption, the viralinoculum is replaced by a fresh culture medium.

The cells infected with AcMNPV, AcRARαP10, AcRARγP10, AcRARαΔP10 andAcRARγΔP10, respectively, were harvested 48 hours after infection,washed in cold PBS buffer, then resuspended in sample buffer and broughtto the boil for 5 min. The same preparation is performed on uninfectedcells.

The total proteins of each preparation are analysed by SDS-PAGE on a 12%polyacrylamide gel. The gels are stained with Coomassie blue.

Comparison of the electrophoretic profiles of the total proteins ofcells infected with the RAR recombinants (AcRARαP10 and AcRARγP10) withthe electrophoretic profile of total proteins of cells infected with thewild-type AcMNPV baculovirus shows that a protein of apparent molecularweight 10 kDa is overproduced in the cells infected with the RARrecombinants. This overproduction is especially large in the cellsinfected with the recombinant AcRARγP10. The band of apparent molecularweight 10 kDa is not seen in the profile of the total proteins of cellsinfected with recombinants AcRARαΔP10 and AcRARγΔP10.

To verify whether the above observations reflect an increase intranscription of the P10 gene, the total cytoplasmic RNAs were isolatedfrom the infected cells 48 h and 72 h after infection, and analysed bydot-blot hybridization using P32-labelled sic! RNA probes complementaryto the P10 and 39K sequences and obtained using the PROMEGA RIBOPROBEkit (PROMEGA, France).

Counting is carried out in a scintillation counter.

The results are illustrated in Table I below, which relates to thevectors depicted in FIGS. 4A and 4B.

                  TABLE I    ______________________________________    cpm    P10                  39K    Virus  48 h       72 h       48 h    72 h    ______________________________________    None     370 ± 10                       1010 ± 40                                  390 ± 12                                          530 ± 25    AcMNPV  32900 ± 580                       26290 ± 450                                 7390 ± 370                                          6050 ± 310    AcD3    31450 ± 800                       37970 ± 720                                 7580 ± 420                                          8520 ± 470    RARα1            73920 ± 1300                       96740 ± 880                                 8840 ± 450                                         11980 ± 630    RARα2            79530 ± 1500                       93120 ± 950                                 9810 ± 600                                         11500 ± 600    RARΔp10            10950 ± 550                       11200 ± 600                                 9550 ± 720                                          5770 ± 220    RARγ1            98290 ± 1100                      146230 ± 1200                                 5300 ± 350                                          5820 ± 280    RARγ2           138090 ± 1450                      142000 ± 1380                                 6080 ± 290                                          6400 ± 300    ______________________________________

Examination of the mean values measured 48 h after infection revealsthat the amount of P10 mRNA produced by the recombinants AcRARαP10 andAcRARγP10 is twice and four times as high, respectively, as thatobserved with the wild-type baculovirus or with the baculovirus AcD3bereft of the polyhedrin promoter. The results obtained 72 hours afterinfection are very similar to those observed 48 hours after infection.At the same time, no significant difference is observed between thedifferent vectors as regards the production of 39K mRNA, measured by wayof an internal control.

EXAMPLE 3 EXPRESSION OF THE CAT REPORTER GENE UNDER THE CONTROL OF THEP10 PROMOTER

The baculoviruses AcP10CAT16, AcP10CAT150 and AcP10CAT230 contain theCAT reporter gene at position +16, +150 and +230, respectively, in thesequence coding for P10, and the gene coding for polyhedrin under thecontrol of its own promoter; the baculoviruses AcRARαCAT16, AcRARγCAT16,AcRARαCAT150, AcRARγCAT150, AcRARαCAT230 and AcRARγCAT230 contain theCAT reporter gene at position +16, +150 and +230, respectively, in thesequence coding for P10, and, in addition, the gene coding for an α or γretinoic acid receptor under the control of the polyhedrin promoter.These different vectors are obtained as described in Example 1 above.

The presence of the CAT protein and also CAT activity are tested for onthe cell extracts prepared from Sf9 cells infected with these differentvectors and harvested 48 hours after infection.

Expression of the CAT gene is determined after immunoelectrophoretictransfer: the total proteins are analysed by SDS-PAGE as described inExample 1 above and transferred onto a nitrocellulose membrane. CAT isdetected with anti-CAT antibodies (5 PRIME>3 PRIME INC.), or bymeasurement of the CAT activity according to the standard methoddescribed by GORMAN et al. Mol. Cell. Biol. No. 2, 1044-1051 (1982)lacuna!. The acetylated forms of the substrate (Ac-CM) are separatedfrom the native substrate (CM) by ascending thin-layer chromatography onsilica.

In the case of cells infected with the vectors of type +150 and +230 andcoexpressing a gene coding for an α or γ retinoic acid receptor, it isestablished by immunotransfer that, for the viruses AcRARαCAT150,AcRARγCAT150, AcRARαCAT230 and AcRARγCAT230, that there is a very markedincrease in the signal corresponding to CAT relative to the virusesAcP10CAT150 and AcP10CAT230. The observed results are confirmed by themeasurement of the CAT activity.

    __________________________________________________________________________    #            SEQUENCE LISTING    - <160> NUMBER OF SEQ ID NOS: 3    - <210> SEQ ID NO 1    <211> LENGTH: 9    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    #Sequence:PromoterATION: Description of Artificial    #or T sequence; n is A or C or G    - <400> SEQUENCE: 1    #          9    - <210> SEQ ID NO 2    <211> LENGTH: 14    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    #Sequence:PromoterATION: Description of Artificial          sequence    - <400> SEQUENCE: 2    #     14           ttca    - <210> SEQ ID NO 3    <211> LENGTH: 14    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    #Sequence:PromoterATION: Description of Artificial    #or T sequence; n is A or C or G    - <400> SEQUENCE: 3    #     14           ttca    __________________________________________________________________________

We claim:
 1. A method for regulating the expression of a gene ofinterest, wherein said method comprises:a) providing an expressionvector comprising a baculovirus promoter and a RARE-type DNA sequencesituated in proximity thereof, and placing said gene of interest underthe transcriptional control of said baculovirus promoter, and b)expressing said gene of interest in the presence of the translationproduct of a gene coding for a RAR-type hormone receptor.
 2. A methodaccording to claim 1, wherein said baculovirus promoter is the P10promoter on the polh promoter.
 3. A method according to claim 1, whereinsaid RAR-type hormone receptor is selected from the group consisting ofRARα receptors, RARβ receptors and RARγ receptors.
 4. A method accordingto claim 1, wherein said RARE-type DNA sequence has the sequence of SEQID NO:
 1. 5. An expression vector, suitable for use in the method ofclaim 1, wherein said vector comprises:a) a first baculovirus promoterand a RARE-type DNA sequence situated in proximity thereof, and b) asecond baculovirus promoter, and a DNA sequence coding for an RAR typehormone receptor, placed under the transcriptional control thereof. 6.An expression vector, wherein said vector comprises at least abaculovirus promoter and a RARE-type DNA sequence situated in proximitythereof, andi) said vector does not comprise the P10 promoter and theRARE-type DNA sequence GTTGACAGTGTTCA SEQ ID NO:2, in the same relativelocation as in the wild P10 gene, or ii) said vector does not comprisethe P10 promoter and the RARE-type DNA sequence GTTGACAGTGTTCA SEQ IDNO:2 separated by the same sequence as in the wild P10 gene.
 7. Anexpression vector according to claim 6, further comprising a gene ofinterest under transcriptional control of said baculovirus promoter. 8.An expression vector according to claim 6, further comprising at leastone site for inserting a gene of interest under transcriptional controlof said baculovirus promoter.
 9. An expression vector according to claim8, further comprising a gene of interest under transcriptional controlof said first baculovirus promoter.
 10. An expression vector accordingto claim 8, further comprising at least one site for inserting a gene ofinterest under transcriptional control of said baculovirus promoter. 11.An expression vector for expressing a gene of interest under thetranscriptional control of the baculovirus polh promoter, wherein saidvector comprises the whole sequence of the baculovirus polh promoter andof the baculovirus P10 promoter, and is devoid of RARE-type sequences inproximity to the P10 promoter.
 12. An expression vector according toclaim 11, which is devoid of the whole P10 coding sequence.
 13. Anexpression vector of claim 11, further comprising at least one site forinsertion of a gene of interest under transcriptional control of thepolh promoter.
 14. An expression vector of claim 11, further comprisinga gene of interest under transcriptional control of the polh promoter.